ABSTRACT Diabetes and its complications still claim the lives of millions of people despite continuing advances in insulin delivery technology primarily because insulin fails to achieve perfect glycemic control. On the other hand, beta cell replacement therapies including vascularized pancreas and isolated islet transplantation are able to fully restore normoglycemia, achieve insulin-independence and can delay end-organ complications. However, these latter therapies suffer from two key limitations, the shortage of organs and the need for life- long immunosuppression to prevent allograft rejection. Furthermore, the intrahepatic portal vein islet transplantation site used in humans is far from ideal and many islets are lost after implantation. An ideal beta cell replacement therapy strives towards both generating an abundant supply of functional beta cells and identifying a minimally invasive, well-vascularized, retrievable site for transplantation that is clinically applicable. After years of research it is now well established that human pluripotent stem cells (hPSCs) can be directed to differentiate into highly enriched physiological functional islet-like clusters (ILCs) in vitro that are capable of curing diabetes in mice. The extracellular matrix (ECM) is a critical component of the cellular niche that helps maintain cellular differentiation and provides tissue-specific signals to guide the fate and behavior of cells. Recent progress in the decellularization of organs has spurred great interest in using natural matrix for regenerative medical applications; yet, few studies have focused on the pancreas in general and the human pancreas to date has not been effectively decellularized and studied. Appreciating the importance of tissue-specific ECM, we have established effective techniques for the decellularization and delipidization of human pancreas tissue to produce several types of natural matrix constructs, including intact 3D matrix, molded sponge scaffolds and a spontaneous gelling hydrogel (hP-ECM). With the challenges of identifying a clinically applicable transplant site that provides for immediate and sufficient oxygen and nutrient delivery, we believe there is compelling rationale to take advantage of the proven proangiogenic and anti-inflammatory properties of ECs and MSCs. Thus, transplanting ILCs with hPSC-derived endothelial cells (ECs) and hPSC-derived mesenchymal stromal cells (MSCs), each providing essential properties, combined with hP-ECM into a prevascularized deviceless retrievable subcutaneous site might provide a more optimal transplant platform. Now, based on this innovative technology we aim to obtain a better understanding of the composition and function of natural hP-ECM in the context of hPSC differentiation to beta cells. The immediate objectives are to characterize human pancreatic extracellular matrix and to use this natural matrix in combination with stem cell-derived ? cells, ECs and MSCs to reconstruct endocrine tissue capable of glucose- stimulated insulin-secretion after transplantation to mice. Our specific aims are to: 1) Comprehensively characterize the human pancreatic and islet ECM proteome, or matrixome, and compare the matrixome of different developmental ages using advanced quantitative mass spectrometry methods in collaboration with Dr. Linjun Li, 2) Construct a hP-ECM - cellular composite tissue graft combining hPSC-ILCs with ECs +/- MSCs and test its function in an immunodeficient murine diabetes model. Ultimately, we envision a bioengineered composite endocrine organ as a highly innovative regenerative medicine strategy for producing potentially autologous insulin-producing tissue for transplantation. These basic enabling studies are the first steps towards developing an effective, minimally invasive transplant platform that is available for all patients with diabetes.